Identification and quantification of constituents of Gardenia jasminoides Ellis (Zhizi) by HPLC-DAD-ESI-MS.

A simple, rapid and specific HPLC method was carried out for the analysis of characteristic constituents in Gardenia jasminoides Ellis (Zhizi), namely iridoids, caffeoyl quinic acid derivatives and crocins. The separation was successfully obtained using a C(18) column by gradient elution with mixtures of methanol and water as mobile phases; detection wavelength was set at 240 nm for iridoid glycosides, 315 nm for quinic acid derivatives and 438 nm for crocins. The analytical method was validated and the quantification of active compounds, namely iridoids, was performed. Linearity, precision, repeatability, stability, accuracy, limit of detection (LOD) and limit of quantification (LOQ) were also reported. This assay was successfully applied for qualitative and quantitative analysis of five commercial samples of G. jasminoides Ellis.

Cyclodextrins as carriers for kavalactones in aqueous media: spectroscopic characterization of (S)-7,8-dihydrokavain and beta-cyclodextrin inclusion complex.

Kavalactones represent the active constituents of kava-kava (Piper methysticum G. Forster), endowed with sedative and anaesthetic properties. Kavalactones are polar constituents, but poorly soluble in water with a low bioavailability. In this study, the formation of inclusion complexes of one of the most representative kavalactone isolated from kava-kava extract, (S)-7,8-dihydrokavain (DHK), with beta-cyclodextrin (beta-CyD) was investigated mainly by spectroscopic methods. NMR experiments were extensively used for the complete characterization of the complex and included (1)H NMR complexation shifts analysis, (1)H NMR diffusion measurements (DOSY), and ROESY experiments. In particular DOSY experiments demonstrated that in the presence of beta-CyD the translational diffusion of kavalactone is sizably slowed down (2.5x10(-10)m(2)/s) with respect to the free drug (4.4x10(-10)m(2)/s) according to the inclusion of DHK in the cavity of (beta-CyD). ROESY experiments confirmed the inclusion of DHK in the hydrophobic pocket of beta-CyD through the primary hydroxyl rim, being the most relevant interactions between the H3' of beta-CyD and the ortho protons on the phenyl ring of the DHK, and between H5' of beta-CyD and the meta/para protons of DHK phenyl ring. The inclusion of the phenyl ring of DHK, leaving the lactone moiety outside of CyD was also confirmed by the induced CD effects. The binary solution DHK/beta-CyD shows a 50% intensity increase of the negative band of the pi-pi* transitions of the phenyl ring with respect to the absorption observed with DHK alone. Molecular dynamics simulations results corroborated and further clarify observed spectroscopic data. It was found that the phenylethyl substituent at C6 has a preferential equatorial position in the free state, and an axial one in the complex, justifying the large downfield shift experienced by H6 of DHK upon binding. Finally the influence of beta-CyD on water solubility of DHK was investigated by phase-solubility studies. In the range 2-4mM of host, solubility of DHK was increased only two-fold, but being beta-CyD also a penetration enhancer, in vivo studies will be performed to clarify a possible role of the complex on the bioavailability of DHK.

Pharmacological in vivo test to evaluate the bioavailability of some St John's Wort innovative oral preparations.

In this study, the optimisation of biopharmaceutical properties of a dried commercial extract of St John's Wort were evaluated employing the in vivo forced swimming test (FST). Three new dosage forms containing beta-cyclodextrin and surfactants (SDS, ASC8) were compared in the FST with the commercial extract. The commercial extract showed antidepressant activity in mice after 60 min at a dosage of 100 mg/kg. The same antidepressant activity appeared in 30 min with a micellar solution of SDS containing the same quantity of extract (100 mg/kg), while with micelles of ASC8 the effect appeared at 15 min and with a dosage of 30 mg/kg. In the case of beta-cyclodextrin the best results were obtained at 30 min, administering 60 mg/kg of the extract. Finally, the influence of the formulations on the water solubility of the constituents of the extract is reported. The tensides dramatically enhanced solubility, in particular that of the more lipophilic compounds, in the case of beta-cyclodextrin this effect was very pronounced for flavonoids and biapigenin, lower for hypericins and practically insignificant for hyperforins.

Analysis and stability of the constituents of St. John's wort oils prepared with different methods.

St. John's wort is a medicinal plant with a long history of use in traditional medicine all over Europe. Traditional preparations and in particular the infused oil from SJW flowers remains one of the most popular and curative topical remedy against ulcerations and burns. The presence of the characteristic polyprenylated acylphloroglucinol derivatives, namely hyperforin and analogs are instead related to the oil's therapeutic activity. Indeed, it is well known that hyperforin has a potent antibacterial activity. In this study we tried to rationalize the production system of the oily preparation in order to obtain the highest concentration and stability of phloroglucinols. Five different samples of SJW oils were evaluated by HPLC-DAD-MS analysis to verify the variability and stability of the constituents according to the following factors: different harvesting time, fresh or dried plant material, use of sunlight or heating systems during extraction. The stability of these oils during 1 year was also tested.

Simultaneous analysis of artemisinin and flavonoids of several extracts of Artemisia annua L. obtained from a commercial sample and a selected cultivar.

Artemisia annua L. (Qinghao) is a promising and potent antimalarial herbal drug. This activity has been ascribed to its component artemisinin, a sesquiterpene lactone that is very effective against drug-resistant Plasmodium species with a low toxicity. Our studies indicate that several flavonoids of A. annua can promote and enhance the reaction of artemisinin with hemin. These data are in good agreement with previous investigations on the in vitro potentiation of antimalarial activity of artemisinin by such flavonoids. As a consequence, in view of a possible use of the phytocomplex rather than pure artemisinin, an HPLC/DAD/MS method is proposed for the simultaneous detection and quantification of both flavonoids and artemisinin. Different extracts, obtained from two different herbal drugs, a commercial sample and a selected cultivar, were analyzed in order to determine which solvents provide the best yields of both artemisinin and flavonoids. Qualitative and quantitative results obtained using an HPLC method are described, which will be useful for developing highly effective herbal drug preparations.

Evaluation of skin permeability of sesquiterpenes of an innovative supercritical carbon dioxide Arnica extract by HPLC/DAD/MS.

Recently, a supercritical carbon dioxide dried extract of Amica flower, with a very high sesquiterpene content was developed. In view of using this extract in formulations for cutaneous application, the ability of sesquiterpenes to permeate the skin was evaluated by HPLC/DAD/MS using the following permeation enhancers: oleic acid (OA), dimethylsulfoxide (DMSO), lauroglycol, isopropyl myristate and Tween 80. A skin permeation study was performed using a modified Franz diffusion cell and the human stratum corneum and epidermis as membrane. Solutions of the enhancers were directly analysed after dilution with methanol or DMSO. A simple RP-HPLC-DAD-MS method for the quantification of the sesquiterpenes was developed and the method showed no interference with the other substances extracted from the skin and the permeation enhancers. The study evidenced that among the selected skin permeation enhancers, DMSO and OA canbe considered as good candidates to be used in preparations for cutaneous application.

Analysis and stability of the constituents of artichoke and St. John's wort tinctures by HPLC-DAD and HPLC-MS.

In continuing our investigations on tinctures, which represent both herbal drug preparations and herbal medicinal products, 40% and 60% v/v tinctures of artichoke and St. John's wort were investigated. Artichoke is largely used in hepatic disorders, while St. John's wort is an anti-inflammatory, antidepressant, and healing agent. Both herbal drugs contain various constituents, although the compounds responsible for the main effects have not yet been completely identified. However, caffeoylquinic acids and flavones seem to be of crucial importance for the activity of artichoke, as well as flavonoids, naphthodianthrones, and phloroglucinol derivatives for St. John's wort, and they are used as marker constituents. Thus, quantification of all these constituents was performed using high-performance liquid chromatography-diode array detection (HPLC-DAD) and HPLC--mass spectrometry (MS) analyses with rutin as external standard. In addition the stability of the constituents of these tinctures from accelerated and long-term testing was also evaluated. From the results it was evidenced that constituent content depends on the solvent used for the extraction. The stability was also shown to be very different and seems to be related to the water content of the tinctures.

Variability in the content of the constituents of Hypericum perforatum L. and some commercial extracts.

Seven samples of Hypericum perforatum L. (St. John's wort) were collected throughout Tuscany; the dried extracts were assayed to determine the concentration of the constituents. Total flavonol content ranged from 4.58% to 15.90%; hypericins ranged from 0.05% to 0.11%; and hyperforins ranged from 1.37% to 20.80%. In addition, four commercially dried extracts were analyzed using the same high-performance liquid chromatographic (HPLC) method; their flavonol contents varied from 10.64% to 15.01%, hypericins varied from 0.03% to 0.20%, and hyperforins varied from 1.18% to 6.54%. The aim of this investigation was to evaluate the contents of the different constituents depending on environmental factors and drying and storage conditions of the wild samples. In addition, the contents of the constituents of the products available to the consumer that were related to quality and the relation of this to safety and efficacy were also evaluated.

Analysis of plant complex matrices by use of nuclear magnetic resonance spectroscopy: St. John's wort extract.

The efficiency of two-dimensional homonuclear (1)H--(1)H correlated spectroscopy and two-dimensional reverse heteronuclear shift correlation spectroscopy (i.e., heteronuclear multiple quantum correlation) in characterizing and evaluating the relative content of herbal extract constituents is demonstrated. These experiments are able to fully assign the proton and carbon resonances of all three classes of constituents present in dried commercial extract of St. John's wort, that is, flavonols, phloroglucinols, and naphthodianthrones, with particular regard to the very unstable phloroglucinols. In addition, shikimic and chlorogenic acids, sucrose, lipids, polyphenols, and traces of solvents of the extractive process (methanol) were also identified. These experiments can be considered to be a very simple and fast analytical method for determining the quality and stability of the titled commercial extract. They represent a generally applicable technique for a rapid screening and a specific measurement of other commercial phytochemicals or, in selected cases, an alternative to the classical analytical techniques such as high-performance thin-layer chromatography, high-performance liquid chromatography, capillary gas chromatography, and electrophoresis.

Evaluation of chemical stability of St. John's wort commercial extract and some preparations.

Thermal and photostability of a commercial dried extract and capsules of St. John's wort (Hypericum perforatum L.) were evaluated under the ICH test conditions. The extract was considered as drug substance and its preparations as drug products. In addition, capsules of different colours corresponding to different opaficient and pigment contents were also evaluated as primary package of drug product and the tests in the secondary pack were performed with amber containers, as well. A selective high-performance liquid chromatography (HPLC) for determination of stability of all the characteristic constituents, namely flavonols, hyperforins and hypericins, was carried out. Photostability testing showed all the constituents to be photosensitive in the tested conditions. However, different opaficients and pigments present in the capsules influenced the stability of the different classes of constituents. Amber containers suggested as secondary packages influenced only in part the photostability of the investigated constituents. Long-term thermal stability testing showed a very low (less than 4 months) hyperforins and hypericins t(90), even if ascorbic and citric acids were added to the formulation. From the results we have obtained it is clear that for St. John's wort preparations, a mere translation of the ICH guidelines to the field of herbal products, as suggested by the WPHMP of the EMEA, cannot be accepted. A revision and adaptation of the storage conditions should be elaborated.

Induction of erythroid differentiation of human K562 cells by 3-O-acyl-1,2-O-isopropylidene-D-glucofuranose derivatives.

In this paper we report the synthesis of twelve 3-O-acyl-1,2-O-isopropylidene-D-glucofuranose derivatives and the results obtained on their effects in inducing erythroid differentiation of human leukemic K562 cells. The data obtained demonstrate that two of the newly synthetized compounds are able to induce erythroid differentiation of K562 cells. In addition, these same compounds potentiate K562 erythroid differentiation induced by cytosine arabinoside, retinoic acid and mithramycin. Inducers of erythroid differentiation stimulating fetal gamma-globin synthesis could be considered for possible use in the experimental therapy of hematological diseases associated with a failure in the expression of adult beta-globin genes.